Cellular function relies on coordinating the thousands of reactions that simultaneously take place within the cell. Cells accomplish this task in large part by spatio-temporally controlling these reactions using diverse intracellular organelles. In addition to classic membrane-bound organelles such as secretory vesicles, mitochondria and the endoplasmic reticulum, cells harbor a variety of membrane-less organelles. Most of these are abundant in both RNA and protein, and are referred to as ribonucleoprotein (RNP) bodies. Among dozens of examples include nuclear bodies such as nucleoli, Cajal bodies, and PML bodies, and cytoplasmic germ granules, stress granules and processing bodies ((Mao et al., 2011), (Anderson and Kedersha, 2009), (Buchan and Parker, 2009), (Handwerger and Gall, 2006)). By impacting a number of RNA processing reactions within cells, these structures appear to play a central role in controlling the overall flow of genetic information, and are also increasingly implicated as crucibles for protein aggregation pathologies ((Li et al., 2013), (Ramaswami et al., 2013)).
From a biophysical standpoint, these structures are remarkable in that they have no enclosing membrane and yet their overall size and shape may be stable over long periods (hours or longer), even while their constituent molecules exhibit dynamic exchange over timescales of tens of seconds (Phair and Misteli, 2000). Moreover, many of these structures have recently been shown to exhibit additional behaviors typical of condensed liquid phases. For example, P granules, nucleoli, and a number of other membrane-less bodies will fuse into a single larger sphere when brought into contact with one another ((Brangwynne et al., 2009), (Brangwynne et al., 2011), (Feric and Brangwynne, 2013)), in addition to wetting surfaces and dripping in response to shear stresses. These observations have led to the hypothesis that membrane-less organelles represent condensed liquid states of RNA and protein that assemble through intracellular phase separation, analogous to the phase transitions of purified proteins long observed in vitro by structural biologists ((Ishimoto and Tanaka, 1977), (Vekilov, 2010)). Consistent with this view, RNP bodies and other membrane-less organelles appear to form in a concentration-dependent manner, as expected for liquid-liquid phase separation ((Brangwynne et al., 2009), (Weber and Brangwynne, 2015), (Nott et al., 2015), (Wippich et al., 2013), (Molliex et al., 2015)). These studies suggest that cells can regulate membrane-less organelle formation by altering proximity to a phase boundary. Movement through such an intracellular phase diagram could be accomplished by tuning concentration or intermolecular affinity, using mechanisms such as posttranslational modification (PTM) and nucleocytoplasmic shuttling.
Recent work has begun to elucidate the molecular driving forces and biophysical nature of intracellular phases. Weak multivalent interactions between molecules containing tandem repeat protein domains appear to play a central role ((Li et al., 2012), (Banjade and Rosen, 2014)). A related driving force is the promiscuous interactions (e.g. electrostatic, dipole-dipole) between segments of conformationally heterogeneous proteins, known as intrinsically disordered protein or intrinsically disordered regions (IDP/IDR, which are typically, although not necessarily, also low complexity sequences, LCS). Hereinafter, the terms intrinsically disordered protein, intrinsically disordered region, and intrinsically disordered protein region are used interchangeably. RNA binding proteins often contain IDRs with the sequence composition biased toward amino acids including R, G, S, and Y, which comprise sequences that have been shown to be necessary and sufficient for driving condensation into liquid-like protein droplets ((Elbaum-Garfinkle et al., 2015), (Nott et al., 2015), (Lin et al., 2015)). The properties of such in vitro droplets have recently been found to be malleable and time-dependent ((Elbaum-Garfinkle et al., 2015), (Zhang et al., 2015), (Weber and Brangwynne, 2012), (Molliex et al., 2015), (Lin et al., 2015), (Xiang et al., 2015), (Patel et al., 2015)), underscoring the role of IDR/LCSs in both liquid-like physiological assemblies and pathological protein aggregates.
Despite these advances, almost all recent studies rely primarily on in vitro reconstitution, due to a lack of tools for probing protein phase behavior within the living cellular context. However, a growing suite of optogenetic tools have been developed to control protein interactions in living cells. The field has primarily focused on precise control over homo- or hetero-dimerization ((Toettcher et al., 2011), (Kennedy et al., 2010), (Levskaya et al., 2009)). But recent work suggests the potential of optogenetics for studying intracellular phases, demonstrating that light-induced protein clustering can be used to activate cell surface receptors (Bugaj et al., 2013), as well as to trap proteins into inactive complexes ((Lee et al., 2014), (Taslimi et al., 2014)).
To date, no platform has been provided which can be used to dynamically modulate intracellular protein interactions, enabling the spatiotemporal control of phase transitions within living cells. Such a platform would be highly desirable.